Control of continuous polyhydroxybutyrate synthesis using calorimetry and flow cytometry

Abstract

The substrate-carbon flow can be controlled in continuous bioreactor cultures by the medium composition, for example, by the C/N ratio. The carbon distribution is optimal when a maximum fraction flows into the desired product and the residual is just sufficient to compensate for the dilution of the microbial catalyst. Undershooting of the latter condition is reflected immediately by changes in the Gibbs energy dissipation and cellular states. Two calorimetric measurement principles were applied to optimize the continuous synthesis of polyhydroxybutyrate (PHB) by Variovorax paradoxus DSM4065 during growth with constantly increasing supply rates of fructose or toxic phenol. Firstly, the changed slope of the heat production rate in a complete heat balanced bioreactor (CHB) indicated optimum carbon channeling into PHB. The extent of the alteration depended directly on the toxic properties of the substrate. Secondly, a flow through calorimeter was connected with the bioreactor as a "measurement loop." The optimum substrate carbon distribution was indicated by a sudden change in the heat production rate independent of substrate toxicity. The sudden change was explained mathematically and exploited for the long-term control of phenol conversion into PHB. LASER flow cytometry measurements distinguished between subpopulations with completely different PHB-content. Populations grown on fructose preserved a constant ratio of two subpopulations with double and quadruple sets of DNA. Cells grown on phenol comprised a third subpopulation with a single DNA set. Rising phenol concentrations caused this subpopulation to increase. It may thus be considered as an indicator of chemostress.

Biothermodynamics of live cells: a tool for biotechnology and biochemical engineering

“…In more recent times it has been demonstrated that the problem associated with biotechnology cannot only be alleviated by scaling the instrument up to the bench scale, but rather by scaling it down to yield a chip calorimeter [31][32][33][34]. The principle of this device, shown in Figure 5, consists in measuring either the adiabatic temperature increase that results from a reacting mixture in a sample injected into the measuring cell, or the steadystate temperature during continuous flow of sample as compared with the temperature obtained from a continuous flow of reference sample.…”

Using isothermal titration calorimetry to real-time monitor the heat of metabolism: A case study using PC12 cells and Aβ(1–40)

“…Recently, a growing number of studies have succeeded in monitoring the heat dissipation of microbial during fermentation by ITC [33][34][35][36][37]. Kemp and his colleagues [23,24] had combined a bioreactor with a flow microcalorimeter to monitor the heat flow rate of the growth of animal cells under the controlled conditions for long cultivation time.…”

From Biochemistry to Physiology: The Calorimetry Connection

“…Liquid cultures in flow calorimeters can easily deplete the dissolved oxygen or achieve toxic concentrations of products in the flow line between the culture vessel and the calorimetric vessel [48]. However, use of a flow loop from a bioreactor to a flow calorimeter can be of great value for bioprocess control [54]. When a gas is flowed through or over the system to maintain gas concentrations, great care must be taken to match the water vapor pressure in the input gas to the water vapor pressure over the culture, tissue, or organism.…”

“…High-pressure ampules are available for most temperature-scanning calorimeters for determination of the effect of pressure on metabolism. Some large volume ( ‡1 l) calorimeters can be instrumented as chemostats for growth of microorganisms in liquid culture, or a regular chemostat can be connected via a flow line to a calorimeter [48,54]. Calorimeters suitable for small animals such as mice and rats are not included in this discussion, but are easily constructed [62].…”